Recent structural studies reveal that dihydropteridine reductase (DHPR) is a member of a large family of dinucleotide requiring enzymes comprising the short-chain dehydrogenases and carbohydrate epimerases. The known members of the differing groups have less than 20% sequence identity yet fold similarly. Each protein contains a glycine rich beta-alpha-beta-fold that can accommodate the AMP component of the dinucleotide cofactor and each contains a Y(Xaa)/3K motif in the vicinity of the active site. In fact, a total of at least five conserved amino acids occur throughout the family and appear essential to the creation of an environment in which a hydride transfer can occur. Three distinct reactions are catalyzed; reduction; oxidation, and epimerisation. In each case a polarized double bond such as C=N or C=O is created or reduced. Using specific site- directed mutagenesis and X-ray crystallography, this proposal intends to determine what structural features are necessary to allow such a group of sequentially disparate proteins to formulate apparently similar structures yet retain sufficient identity that three differing reactions can occur. A unique hypothesis common to each of the dinucleotide requiring enzyme mechanisms is that the tyrosine/lysine motif could be a novel method for facilitating proton exchange with the tyrosine phenolic group (pK about 10). Specific mutational and kinetic experiments that aim to confirm this concept are described for both DHPR and the 2,3-dihydro-2,3- dihydroxybenzoate dehydrogenase (DDBDH), which is to be purified, crystallized and structurally characterized. It is anticipated that the experimental results will demonstrate that structural folding patterns that ensure conserved amino acids retain a characteristic function are equally or more important than sequence when looking for protein behavioral patterns. As a corollary to the investigation it is also intended to derive the structure of the Leishmania ltdh encoding resistance protein, as it demonstrates unusual sequence similarities to the above family of enzymes.